The projects center on experiments designed to obtain information about the mechanism and regulation of ribosome biosynthesis in eukaryotes. The lower eukaryote, Neurospora crassa, will be used as the model organism since it is highly amenable to the conbined genetical and biochemical approaches to be employed. Four projects will be carried out (the analytical techniques are given in parentheses): (1) Three temperature-sensitive mutants of Neurospora crassa have been obtained which have conditional defects in cytosolic ribosome biosynthesis. Each of the mutant strains accumulates disproportionate amounts of the two ribosomal subunits at the nonpermissive temperatures. Here, experiments will investigate whether the underaccumulation of one of the high-molecular-weight ribosomal RNAs (rRNA) is accompanied by repressed ribosomal protein synthesis for the relevant subunit, or whether ribosomal proteins are made normally and then are stored or degraded (RNA, ribosomal protein isolation; acrylamide gel electrophoresis); (2) A strain has been obtained which has two nucleolar organizer regions. Experiments will be done to confirm that twice the wild-type number of rRNA genes are present in the strain and to investigate whether mechanisms exist to decrease ("demagnify") the number of rRNA genes to the wild-type level. In addition the effect of extra rRNA genes on precursor-rRNA synthesis and processing will be examined (RNA isolation; acrylamide gel electrophoresis; nuclear isolation; DNA-RNA hybridization); (3) Mutant strains have been obtained that are simultaneously cold-sensitive and cycloheximide-sensitive. Since cycloheximide is known to act on the large ribosomal subunit, we shall investigate whether or not an altered ribosomal structural component (possibly a ribosomal protein) can be found in the mutant strains compared with the wild-type (ribosomal protein analysis; in vitro protein synthesis); (4) New cold-sensitive mutants of Neurospora will be isolated and these will be screened for conditional defects in ribosome biosynthesis. Any such mutants will be studied further to pinpoint the molecular event(s) involved (mutagenesis; sucrose gradient analysis of ribosomes).